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Literature summary extracted from

  • Patrick, W.M.; Matsumura, I.
    A study in molecular contingency: glutamine phosphoribosylpyrophosphate amidotransferase is a promiscuous and evolvable phosphoribosylanthranilate isomerase (2008), J. Mol. Biol., 377, 323-336.
    View publication on PubMedView publication on EuropePMC

Application

EC Number Application Comment Organism
2.4.2.14 additional information marginal secondary enzyme activities can dramatically improve the fitness of contemporary organisms Escherichia coli K-12

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.4.2.14 wildtype, mutant I198V and variants 1-04 and 2-02 of mutant I198V in pCA24N for expression as N-terminal hexa-His-tagged, C-terminal GFP-tagged proteins in Escherichia coli JMB9 DELTA TrpF (auxotrophic for tryptophan) and/or Escherichia coli KK8(pDM), mutant I198V in pCDF-1b for error-prone PCR based mutagenesis yielding variant 1-04 Escherichia coli K-12
5.3.1.24 expressed in Escherichia coli XL1-Red cells Escherichia coli

Protein Variants

EC Number Protein Variants Comment Organism
2.4.2.14 I198V by missense mutation A592G, purF(I198V) and variant purF(1-04) in pCA24N by two polymerase error prone PCR strategy (1. mutazyme, 2. Taq DNA polymerase in Thermopol reaction buffer) followed by second directed mutagenesis on purF(1-04) by passaging through Escherichia coli mutator strain XL1-Red leading to variant purF(2-02), mutant I198V and variants 1-04 and 2-02 improved growth of Escherichia coli JMB9 DELTA TrpF (auxotrophic for tryptophan) on medium lacking tryptophan by gained phosphoribosylanthranilate isomerase activity Escherichia coli K-12
2.4.2.14 N328S recurring mutation after error-prone PCR, found in variant 1-04 of mutant I198V which also harbours mutations: I37M, A39T, E85G, P88S, N124S, I198V, K282R and one silent, Asn328 is implicated in PRPP binding and a critical residue for improving phosphoribosylanthranilate isomerase activity. Variant 2-02 has the same open reading frame as 1-04 but optimized transcription. Escherichia coli K-12

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.4.2.14 additional information
-
 phosphoribosylanthranilate variant 1-04, kcat/Km = 0.3 1/s*M, initial velocity measurements over time at substrate concentrations to up to 2 mM Escherichia coli K-12

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.4.2.14 L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O Escherichia coli K-12
-
 5-phospho-beta-D-ribosylamine + diphosphate + L-glutamate
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
2.4.2.14 Escherichia coli K-12 P0AG16
-
-
5.3.1.24 Escherichia coli
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.4.2.14 hexa-His-tagged mutant I198V and its variant 1-04 by metal affinity chromatography in presence of 1 mM beta-mercaptoethanol and 10% (v/v) glycerol, elution with 250 mM imidazole followed by dialysis Escherichia coli K-12
5.3.1.24 Ni-NTA column chromatography Escherichia coli

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.4.2.14 L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
-
 Escherichia coli K-12 5-phospho-beta-D-ribosylamine + diphosphate + L-glutamate
-
 ?
2.4.2.14 phosphoribosylanthranilate 5 µM PurF mutant I198V or variant 1-04, in presence of 0.5 mM beta-mercaptoethanol, pH 8.6, 23 °C, 16 h Escherichia coli K-12 indoleglycerol phosphate + CO2 + H2O Amadori rearrangement of aminoaldose (phosphoribosylanthranilate) to aminoketose (indoleglycerol phosphate) ?
5.3.1.24 N-(5-phospho-beta-D-ribosyl)anthranilate
-
 Escherichia coli 1-(2-carboxyphenylamino)-1-deoxy-D-ribulose 5-phosphate
-
 ?

Synonyms

EC Number Synonyms Comment Organism
2.4.2.14 glutamine phosphoribosylpyrophosphate amidotransferase
-
 Escherichia coli K-12
2.4.2.14 PurF
-
 Escherichia coli K-12
5.3.1.24 PRA isomerase
-
 Escherichia coli
5.3.1.24 PRAI
-
 Escherichia coli
5.3.1.24 PurF(I198V) the glutamine phosphoribosylpyrophosphate amidotransferase mutant I198V possesses detectable PRAI activity Escherichia coli

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.4.2.14 additional information
-
 phosphoribosylanthranilate mutant I198V, modestly higher rate than spontaneous substrate hydrolysis of <1/h Escherichia coli K-12
2.4.2.14 additional information
-
 phosphoribosylanthranilate variant 1-04, 25 to 30-fold more active than mutant I198V but 2.3*10(7)-fold less efficient than phosphoribosylanthranilate isomerase Escherichia coli K-12
2.4.2.14 additional information
-
 phosphoribosylanthranilate variant 1-04, conversion of 5.5% of available substrate, 22 turnovers per active site Escherichia coli K-12